Raman spectroscopy is a spectroscopic technique used to study vibrational, rotational, and other low-frequency modes of materials. The technique relies on inelastic scattering of monochromatic light in the visible, near infrared, or near ultraviolet range. The light interacts with phonons or other excitations in the material, resulting in a shift in the energy of the light photons from which shift information about the phonon modes in the system can be derived. A Raman spectrum contains many different peaks. Each peak corresponds to the energy of the vibration of a chemical bond in the molecule. Therefore, a Raman spectrum can be nearly a unique fingerprint of a molecule. The spectrum can be used, for example, in analytical chemistry to identify the various molecules in an unknown sample.
Typically, in a conventional Raman spectroscopy set up, such as that shown in FIG. 1, a sample 106 is illuminated with a laser beam 102. The laser beam 102 is generated by a laser 100, deflected via a semi-transparent or dichroic mirror 108 through an objective lens 104 onto the sample 106. The radiation which is scattered back from the sample surface 106 travels through the lens 104 in the opposite direction and passes through the semi-transparent mirror 108. The scattered light then passes through other parts of the spectrometer, which are not shown in the drawing, where wavelengths close to the laser line, produced by elastic Rayleigh scattering, are filtered out. The remainder of the collected scattered light is dispersed onto a detector 110.
Conventional Raman spectroscopy is an ideal tool for non-destructive in-situ investigations and is characterized by both its very high spectral resolution, which permits effective discrimination among various species, and by its high spatial resolution (when a microscope is used). Because the inelastic Raman scattering is relatively weak, Raman spectrometers typically use holographic diffraction gratings and multiple dispersion stages to achieve a high degree of rejection of the laser light. However, even with these refinements, the low intensity of normal Raman scattering and the tendency of many analytes to fluoresce under laser excitation have prevented wider application of the technique as a sensitive spectroscopic probe.
In an enhanced Raman technique called Surface Enhanced Raman Spectroscopy (SERS), the weak Raman scattering intensity is greatly strengthened (by a factor of many orders of magnitude as compared to the intensity obtained from the same number of molecules in solution or in the gas phase) by either attaching the molecules which produce the inelastic scattering to metal structures of nanoscale size or locating the metal structures in the vicinity of the molecules. The exact mechanism involved has not been determined at the present time, but the SERS effect has been observed for molecular species adsorbed on rough metal surfaces. See, for example, “Surface Enhanced Raman Scattering”, Chang, R. K.; Furtak, T. E., Plenum, New York, (1982); “Surface Enhanced Raman Scattering; Chemical and Biochemical Applications of Lasers”, Van Duyne, R. P., edited by C. B. Moore, v. 4, p. 101, Academic Press, New York, (1979); “Surface Enhanced Spectroscopy”, Moskovits, M., Review of Modern Physics, v. 57, p. 783 (1985); “Surface Enhanced Raman Scattering; in Spectroelectrochemistry: Theory and Practice”, Birke, R. L., Lombardi, J. R., edited by R. J. Gale, Plenum, (1988). At the same time, the proximity of the molecular species to the surface of the substrate provides a non-radiative pathway for relaxation from the excited states of the molecules, which successfully quenches fluorescence.
The SERS technique can be used to conduct Raman spectroscopy studies of analytes, such as pharmaceutical compounds, and other organic substances that are too fluorescent, or are present in very minute concentrations in their matrices to yield usable Raman spectra. One area of particular interest is the applicability of SERS to natural and synthetic dyes which has great importance in the museum field in support of art historical, archaeological, and anthropological studies, and to evaluate susceptibility of artistic works to light induced fading.
Organic substances have been used for millennia as textile dyes or, complexed with metal ions, as pigments for artistic or utilitarian objects. Anthraquinones such as alizarin and purpurin (from the root of rubia tinctorum L.), carminic acid (from the insect Dactylopius coccus C.), the laccaic acids (from the insect Kerria lacca K.), or naphtoquinones such as lawsone and juglone, neoflavonoids such as brazilein, flavonoids such as quercetin and morin, and the alkaloid berberine, are found in works of art as red, purple, brown, and yellow dyes. These molecules are also found in modern products: carminic acid is a common red dye for beverages and cosmetics. Beyond the arts field, the molecules are all relevant to other fields, either directly or as proxies for other compounds: in pharmaceutical studies (berberine, anthraquinoid anticancer drugs), in the forensic field (in evidence such as lipsticks, inks, drugs of abuse, toxic agents), in the food colorants industry, and in the textile dyeing industry.
The identification of dyes is generally conducted by extraction followed by high performance liquid chromatography analysis. This analysis requires a sizable sample (5 millimeter of threads from a textile, for instance) and makes it impossible to analyze paintings and drawings, where samples larger than 100 μm cannot be removed. However, SERS has been successfully applied to the identification of synthetic anthraquinones, an SERS method has been developed for the extraction and identification of alizarin at sub-nanogram levels in paint samples and positive results have been obtained for highly fluorescent molecules, such as alizarin, purpurin, laccaic acid, carminic acid, kermesic acid, shikonin, juglone, lawsone, brazilin and brazilein, haematoxylin and haematein, fisetin, quercitrin, quercetin, rutin, morin, and berberine.
One prior art technique allows the spectra of natural dyes to be obtained from actual textile samples and directly from extremely small samples (1 mm×25 μm) of dyed fibers with SERS active substrates including Ag colloids obtained by reduction with sodium citrate or with hydroxylamine hydrochloride, silver films obtained by chemical deposition with the Tollens reaction, and silver nanoislands films obtained by vacuum evaporation.
However, it would be desirable to be able to analyze even smaller samples and, particularly, to be able to selectively analyze portions of small samples.